Quantitative Real-time PCR Analysis Research Articles

Downregulation of NAT1 Expression is Associated with Poor Prognosis and Immune Infiltration in COAD.

An increasing corpus of evidence has identified the involvement of N-acetyltransferase 1 (NAT1), a member of the NAT family, in the progression of various cancers. However, the specific function of NAT1 in colon cancer (COAD) remains elusive. This study aims to decip her the role of NAT1 in COAD and its associated mechanisms. The Tumor Immunity Evaluation Resource (TIMER), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) databases were employed to assess the NAT1 expression level in COAD. The differential expression between COAD and normal colon tissue was further validated using quantitative real-time reverse-transcription PCR (RT-qPCR) and Western blot (WB) analyses. Additionally, survival analysis of NAT1 in COAD was carried out using the PrognoScan database and TCGA dataset. The functions of NAT1 were explored through gene set enrichment analysis (GSEA) and immuno-infiltration analysis. There was a significant reduction in NAT1 expression in COAD samples compared to normal tissue. Notably, low NAT1 expression in COAD correlated significantly with various clinical parameters such as tumor stage (T stage, N stage, M stage, pathologic stage), primary therapy outcome, carcinoembryonic antigen (CEA) level, and lymphatic invasion. The downregulation of NAT1 was also strongly linked with poor outcomes in overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS). Cox regression analysis highlighted NAT1 as an independent prognostic indicator for overall survival in COAD patients. GSEA results revealed NAT1's involvement in multiple pathways, including the neuroactive ligand-receptor interaction, olfactory transduction, olfactory signaling, extracellular matrix receptor interaction, calcium signaling, and focal adhesion pathways. Furthermore, NAT1 expression was found to significantly correlate with infiltration levels of various immune cells. The findings reveal NAT1's potential as a valuable prognostic biomarker for COAD. Moreover, its associated mechanisms offer insights that might pave the way for therapeutic interventions for COAD patients.

Identification of WRKY gene family in Dioscorea opposita Thunb. reveals that DoWRKY71 enhanced the tolerance to cold and ABA stress.

WRKY transcription factors constitute one of the largest plant-specific gene families, regulating various aspects of plant growth, development, physiological processes, and responses to abiotic stresses. This study aimed to comprehensively analyze the WRKY gene family of yam (Dioscorea opposita Thunb.), to understand their expression patterns during the growth and development process and their response to different treatments of yam and analyze the function of DoWRKY71 in detail. A total of 25 DoWRKY genes were identified from the transcriptome of yam, which were divided into six clades (I, IIa, IIc, IId, IIe, III) based on phylogenetic analysis. The analysis of conserved motifs revealed 10 motifs, varying in length from 16 to 50 amino acids. Based on real-time quantitative PCR (qRT-PCR) analysis, DoWRKY genes were expressed at different stages of growth and development and responded differentially to various abiotic stresses. The expression level of DoWRKY71 genes was up-regulated in the early stage and then down-regulated in tuber enlargement. This gene showed responsiveness to cold and abiotic stresses, such as abscisic acid (ABA) and methyl jasmonate (MeJA). Therefore, further study was conducted on this gene. Subcellular localization analysis revealed that the DoWRKY71 protein was localized in the nucleus. Moreover, the overexpression of DoWRKY71 enhanced the cold tolerance of transgenic tobacco and promoted ABA mediated stomatal closure. This study presents the first systematic analysis of the WRKY gene family in yam, offering new insights for studying WRKY transcription factors in yam. The functional study of DoWRKY71 lays theoretical foundation for further exploring the regulatory function of the DoWRKY71 gene in the growth and development related signaling pathway of yam.

ACE2 Rescues Sepsis-Associated Encephalopathy by Reducing Inflammation, Oxidative Stress, and Neuronal Apoptosis via the Nrf2/Sestrin2 Signaling Pathway.

Neuroinflammation and oxidative stress contribute to the progression of sepsis-associated encephalopathy (SAE). Angiotensin-converting enzyme 2 (ACE2) is considered to be a neuroprotective factor due to its anti-inflammatory and antioxidant properties. However, the role of ACE2 on myeloid cells in regulating SAE and the underlying mechanism warrants further exploration. SAE was induced in ACE2 transgenic (TG), knockout (KO), and bone marrow (BM) chimeric mice by cecal ligation and puncture (CLP). The expression levels of apoptosis-, oxidation- and neuroinflammation-associated mediators and morphological changes were monitored by quantitative real-time PCR analyses and histological examinations in the cortex of septic mice. The contents of angiotensin (Ang) II and Ang-(1-7) along with the activity of ACE2 were examined with commercial kits. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Sestrin2 was detected by immunoblotting analysis. Our results indicated that the expression of cortical ACE2 was significantly reduced in the early phase of CLP-induced sepsis. Moreover, ACE2 overexpression in TG mice conferred neuroprotection against sepsis, as evidenced by alleviated neuronal apoptosis, oxidative stress, and proinflammatory M1-like microglial polarization, accompanied by upregulation of the Ang-(1-7), Nrf2, and Sestrin2 protein levels. Conversely, ACE2 deficiency in KO mice exacerbated SAE. The neuroprotective effects of ACE2 were further confirmed in wild-type mice transplanted with ACE2-TG and KO BM cells. Therefore, our data suggest that myeloid ACE2 exerts a protective role in the pathogenesis of SAE, potentially by activating Ang-(1-7)-Nrf2/sestrin2 signaling pathway, and highlight that upregulating ACE2 expression and activity may represent a promising approach for the treatment of SAE in patients with sepsis.

Fibronectin binds integrin α5β1 to regulate macular neovascularization through the Wnt/β-catenin signaling pathway

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the macula. The formation of macular neovascularization (MNV) and subretinal fibrosis of AMD is the most classic cause of the loss of vision in older adults worldwide. While the underlying causes of MNV and subretinal fibrosis remain elusive, the common feature of many common retinal diseases is changes the proportions of protein deposition in extracellular matrix (ECM) when compared to normal tissue. In ECM, fibronectin (FN) is a crucial component and plays a pivotal part not only in fibrotic diseases but also in the process of angiogenesis. The study aims to understand the role of ligand FN and its common integrin receptor α5β1 on MNV, and to understand the molecular mechanism involved. To study this, the laser-induced MNV mouse model and the rhesus macaque choroid-retinal endothelial cell line (RF/6A) chemical hypoxia mode were established, and the FN-α5β1 expression levels were detected by immunohistochemistry (IHC) and quantitative real-time PCR analysis (qRT-PCR). Fibronectin expression was silenced using small interfering RNA (siRNA) targeting FN. The tube formation and vitro scratch assays were used to assess the ability to form blood vessels and cell migration. To measure the formation of MNV, immunofluorescence, and Western blot assays were used. These results revealed that the expressions of FN and integrin α5β1 were distinctly increased in the laser-induced MNV mouse model and in the RF/6A cytochemically induced hypoxia model, and the expression tendency was identical. After the use of FN siRNA, the tube formation and migration abilities of the RF/6A cells were lower, the ability of endothelial cells to proliferate was confined and the scope of damage caused by the laser in animal models was significantly cut down. In addition, FN gene knockdown dramatically inhibited the expression of Wnt/β-catenin signal. The interaction of FN with the integrin receptor α5β1 in the constructed model, which may act through the Wnt/β-catenin signaling pathway, was confirmed in this study. In conclusion, FN may be a potential new molecular target for the prevention and treatment of subretinal fibrosis and MNV.

Circ_0104652 Promotes the Proliferation and Migration of ox-LDL-Stimulated Vascular Smooth Muscle Cells via Stabilizing ADAMTS7 and HMGB1.

Atherosclerosis (AS) stands as the primary contributor to cardiovascular disease, a pervasive global health concern. Extensive research has underscored the pivotal role of circular RNAs (circRNAs) in cardiovascular disease development. However, the specific functions of numerous circRNAs in AS remain poorly understood. Quantitative real-time PCR analysis revealed a significant upregulation of circ_0104652 in oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cells (VSMCs). Loss-of-function experiments were subsequently employed to assess the impact of circ_0104652 on ox-LDL-induced VSMCs. Silencing circ_0104652 was found to impede the proliferation and migration while promoting the apoptosis of ox-LDL-stimulated VSMCs. Mechanistic assays unveiled that circ_0104652 stabilized ADAM metallopeptidase with thrombospondin type 1 motif 7 (ADAMTS7) and high mobility group box 1 (HMGB1) by recruiting eukaryotic translation initiation factor 4A3 (EIF4A3) protein. Rescue assays further confirmed that circ_0104652 exerted its influence on ox-LDL-induced VSMC proliferation through modulation of ADAMTS7 and HMGB1. This study elucidates the role of the circ_0104652/EIF4A3/ADAMTS7/HMGB1 axis in ox-LDL-stimulated VSMCs, providing valuable insights into the intricate mechanisms involved.

Transcriptional Profiling of BpWRKY49 Reveals Its Role as a Master Regulator in Stress Signaling Pathways in Birch (Betula platyphylla)

The WRKY family of transcription factors (TFs) is one of the most diverse families in plants, playing crucial roles in various plant growth and stress response processes. Asian white birch (Betula platyphylla) is a globally distributed tree species that holds ecological, medical, and economic significance. However, the regulatory mechanisms of WRKY TFs in birch remain poorly understood. Herein, we cloned and characterized the BpWRKY49 gene from birch. Through bioinformatics analyses, we revealed the potential involvement of BpWRKY49 in both biotic and abiotic stress responses. In addition, BpWRKY49 was found to be localized in the nucleus and exhibited transcriptional activity in yeast. Transactivation assays further confirmed that BpWRKY49 exhibited transcriptional activity at its C-terminal end. Notably, our binding specificity assays demonstrated the specific interaction of BpWRKY49 with the W-box cis element in vitro. Furthermore, tissue-specific expression analysis demonstrated that BpWRKY49 exhibited the highest expression level in the roots. Real-time quantitative PCR (RT-qPCR) analysis of birch plants subjected to salt and drought treatments revealed that BpWRKY49 displayed significant 30-fold and 10-fold upregulations under salt and drought stress conditions, respectively. DAP-seq analysis of BpWRKY49 identified a total of 21,832 peaks, with 3477 occurring in the promoter region of genes. Gene ontology (GO) enrichment analysis highlighted prominent terms related to defense against biotic stress, followed by terms associated with abiotic stress and development. Y1H assays of three genes provided evidence for the binding ability of BpWRKY49 to the promoters of BpPUB21, BpBTL15, and BpHIP47 in vitro. Collectively, our findings strongly suggest that BpWRKY49 possesses diverse functions and may activate multiple genes to contribute to various biological processes, including salt stress tolerance, in birch.

Pungency related gene network in Allium sativum L., response to sulfur treatments

Pungency of garlic (Allium sativum L.) is generated from breakdown of the alk(en)yl cysteine sulphoxide (CSO), alliin and its subsequent breakdown to allicin under the activity of alliinase (All). Based on recent evidence, two other important genes including Sulfite reductase (SiR) and Superoxide dismutase (SOD) are thought to be related to sulfur metabolism. These three gene functions are in sulfate assimilation pathway. However, whether it is involved in stress response in crops is largely unknown. In this research, the order and priority of simultaneous expression of three genes including All, SiR and SOD were measured on some garlic ecotypes of Iran, collected from Zanjan, Hamedan and Gilan, provinces under sulfur concentrations (0, 6, 12, 24 and 60 g/ per experimental unit: pot) using real-time quantitative PCR (RT-qPCR) analysis. For understanding the network interactions between studied genes and other related genes, in silico gene network analysis was constructed to investigate various mechanisms underlying stimulation of A. sativum L. to cope with imposed sulfur. Complicated network including TF-TF, miRNA-TF, and miRNA-TF-gene, was split into sub-networks to have a deeper insight. Analysis of q-RT-PCR data revealed the highest expression in All and SiR genes respectively. To distinguish and select significant pathways in sulfur metabolism, RESNET Plant database of Pathway Studio software v.10 (Elsevier), and other relative data such as chemical reactions, TFs, miRNAs, enzymes, and small molecules were extracted. Complex sub-network exhibited plenty of routes between stress response and sulfate assimilation pathway. Even though Alliinase did not display any connectivity with other stress response genes, it showed binding relation with lectin functional class, as a result of which connected to leucine zipper, exocellulase, peroxidase and ARF functional class indirectly. Integration network of these genes revealed their involvement in various biological processes such as, RNA splicing, stress response, gene silencing by miRNAs, and epigenetic. The findings of this research can be used to extend further research on the garlic metabolic engineering, garlic stress related genes, and also reducing or enhancing the activity of the responsible genes for garlic pungency for health benefits and industry demands.

Functional Differentiation of the Duplicated Gene BrrCIPK9 in Turnip (Brassica rapa var. rapa).

Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip (Brassica rapa var. rapa) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The BrrCIPK9 gene in turnip has not been characterized. In this study, two duplicated genes, BrrCIPK9.1 and BrrCIPK9.2, were screened from the turnip genome. Based on the phylogenetic analysis, BrrCIPK9.1 and BrrCIPK9.2 were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that BrrCIPK9.1 and BrrCIPK9.2 have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, BrrCIPK9.1 responded to high-pH (pH 8.5) stress, while BrrCIPK9.2 retained its ancestral function (low K+), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated BrrCIPK9 genes and provide a basis for further functional studies of BrrCBLs-BrrCIPKs in turnip.

Tobacco Transcription Factor NtWRKY70b Facilitates Leaf Senescence via Inducing ROS Accumulation and Impairing Hydrogen Sulfide Biosynthesis.

Leaf senescence is the terminal stage of leaf development, and its initiation and progression are closely controlled by the integration of a myriad of endogenous signals and environmental stimuli. It has been documented that WRKY transcription factors (TFs) play essential roles in regulating leaf senescence, yet the molecular mechanism of WRKY-mediated leaf senescence still lacks detailed elucidation in crop plants. In this study, we cloned and identified a tobacco WRKY TF gene, designated NtWRKY70b, acting as a positive regulator of natural leaf senescence. The expression profile analysis showed that NtWRKY70b transcript levels were induced by aging and hydrogen peroxide (H2O2) and downregulated upon hydrogen sulfide (H2S) treatment. The physiological and biochemical assays revealed that overexpression of NtWRKY70b (OE) clearly promoted leaf senescence, triggering increased levels of reactive oxygen species (ROS) and decreased H2S content, while disruption of NtWRKY70b by chimeric repressor silencing technology (SRDX) significantly delayed the onset of leaf senescence, leading to a decreased accumulation of ROS and elevated concentration of H2S. The quantitative real-time PCR analysis showed that the expression levels of various senescence-associated genes and ROS biosynthesis-related genes (NtRbohD and NtRbohE) were upregulated in OE lines, while the expression of H2S biosynthesis-related genes (NtDCD and NtCYSC1) were inhibited in OE lines. Furthermore, the Yeast one-hybrid analysis (Y1H) and dual luciferase assays showed that NtWRKY70b could directly upregulate the expression of an ROS biosynthesis-related gene (NtRbohD) and a chlorophyll degradation-related gene (NtPPH) by binding to their promoter sequences. Accordingly, these results indicated that NtWYKY70b directly activated the transcript levels of NtRbohD and NtPPH and repressed the expression of NtDCD and NtCYCS1, thereby promoting ROS accumulation and impairing the endogenous H2S production, and subsequently accelerating leaf aging. These observations improve our knowledge of the regulatory mechanisms of WRKY TFs controlling leaf senescence and provide a novel method for ensuring high agricultural crop productivity via genetic manipulation of leaf senescence in crops.

Differential Gene Expression Profiling during Avian Immune Organ Development: Insights from Thymus and Bursa of Fabricius Transcriptome Analysis

Pluripotent hematopoietic stem cells undergo maturation to regenerate blood cells. T-cell and B-cell maturation from common lymphoid progenitors (CLPs) is regulated by distinct transcription factors and signaling pathways. Notch signaling, GATA3, and TCF1 drive T-cell fate, while EBF1, PU.1, E2A, Pax5, TCF1, and Foxo1 regulate B-cell differentiation. Alterations in these factors can cause lineage deviations. Gene expression profiling of the chick thymus and bursa reveals dynamic expression patterns of transcription factors, cytokines, and signal molecules. Notch1 and Dll1 expression that increase during later stages indicate the ongoing role of Notch signaling in T-cell lineage maintenance. The current study aimed to identify differentially expressed genes during the development of the avian immune organs, focusing on the thymus and bursa using 24 Ross 308 avian breed. The mRNA libraries from these organs were analyzed using quantitative Real-time PCR analysis at six time points spanning the embryonic ages (days 15 and 18) and post-hatch age (days 3, 7, 14, and 28). The data for the gene expression indicated significant variations across different stages of immune organ development. Differential gene expression was observed between sorted T and B-cells, with GATA3, CD3e, CD4, and Ptprc showing higher expression in the T-cell population, and Pax5 and CD81 exhibiting higher expression in the B-cell population. Notably, ENO1 and IRF4 showed higher expression in T-cells at E15 and B-cells at E18. The study highlights the importance of regulatory factors and genes in maintaining cellular identity, furthers the understanding of avian immunology, and has the potential for improving poultry health and studying immune-related diseases in humans. These findings pave the way for further research on the role of biochemical components under important disease conditions in avian immunology and their potential applications for poultry health. Keywords: Avian lymphocyte, B-cell maturation, Gene expression, Regulatory elements, T-cell

Identification of potential cell death-related biomarkers for diagnosis and treatment of osteoporosis

BackgroundThis study aimed to identify potential biomarkers for the diagnosis and treatment of osteoporosis (OP).MethodsData sets were downloaded from the Gene Expression Omnibus database, and differentially programmed cell death-related genes were screened. Functional analyses were performed to predict the biological processes associated with these genes. Least absolute shrinkage and selection operator (LASSO), support vector machine (SVM), and random forest (RF) machine learning algorithms were used to screen for characteristic genes, and receiver operating characteristics were used to evaluate the diagnosis of disease characteristic gene values. Gene set enrichment analysis (GSEA) and single-sample GSEA were conducted to analyze the correlation between characteristic genes and immune infiltrates. Cytoscape and the Drug Gene Interaction Database (DGIdb) were used to construct the mitochondrial RNA-mRNA-transcription factor network and explore small-molecule drugs. Reverse transcription real-time quantitative PCR (RT-qPCR) analysis was performed to evaluate the expression of biomarker genes in clinical samples.ResultsIn total, 25 differential cell death genes were identified. Among these, two genes were screened using the LASSO, SVM, and RF algorithms as characteristic genes, including BRSK2 and VPS35. In GSE56815, the area under the receiver operating characteristic curve of BRSK2 was 0.761 and that of VPS35 was 0.789. In addition, immune cell infiltration analysis showed that BRSK2 positively correlated with CD56dim natural killer cells and negatively correlated with central memory CD4 + T cells. Based on the data from DGIdb, hesperadin was associated with BRSK2, and melagatran was associated with VPS35. BRSK2 and VPS35 were expectably upregulated in OP group compared with controls (all p < 0.05).ConclusionsBRSK2 and VPS35 may be important diagnostic biomarkers of OP.

Abstract 202: Spry1 targeting enhances E-cadherin expression in cutaneous melanoma

Abstract Background: Cutaneous melanoma (CM) is a very aggressive malignancy that still represents the deadliest form of skin cancer. About 50% of CM harbors the activating BRAFV600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently reported that Spry1 knockdown (Spry1KO) reduced the expression of several markers of epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Loss of E-cadherin is a hallmark of EMT in CM, and the presence of aberrant E-cadherin expression in CM is usually associated with a significantly worse overall survival. Based on these premises, in this study we wondered to explore whether Spry1 influences EMT through the modulation of E-cadherin expression. Material and Methods: SPRY1 gene was knocked-out using the CRISPR strategy in BRAF wild-type (wt) and in BRAF-mutant CM cell lines. By using in vitro and in vivo models, the effects of Spry1KO on E-cadherin expression and localization was investigated through RNA-sequencing (RNA-seq), quantitative real-time PCR, western blot, and immunofluorescence analyses. To gain insight into Spry1 interactome, immunoprecipitation coupled to mass spectrometry (IP-MS) was performed. Results and Discussion: E-cadherin mRNA and protein levels were significantly increased both in vitro and in vivo in Spry1KO clones. In addition, immunofluorescence analysis revealed a sustained redistribution of E-cadherin to the plasma membrane following Spry1 loss, thus confirming that Spry1 plays a role in EMT in CM. E-cadherin requires p120-catenin (p120) to maintain cell adhesion and functional adherens junctions. Indeed, p120 dysregulation results in the rapid turnover of E-cadherin complexes. Intriguingly, IP-MS analyses identified several Spry1 protein partners, including p120. Although a number of functional studies are still ongoing, preliminary western blot analysis of cytosolic and mitochondrial proteins indicated that Spry1 and p120 were mostly located in mitochondria. Conclusion: Spry1 reduced E-cadherin expression in vitro and in vivo. Starting from preliminary data, we hypothesized that Spry1-p120 interaction might influence E-cadherin distribution over the plasma membrane. Citation Format: Barbara Montico, Giorgio Giurato, Roberto Guerrieri, Annamaria Salvati, Francesca Colizzi, Lorena Baboci, Luca Sigalotti, Alessia Covre, Michele Maio, Agostino Steffan, Tuula Anneli Nyman, Alessandro Weisz, Maurizio Mongiat, Eva Andreuzzi, Elisabetta Fratta. Spry1 targeting enhances E-cadherin expression in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 202.

Mechanism of oxalate decarboxylase Oxd_S12 from Bacillus velezensis BvZ45-1 in defense against cotton verticillium wilt.

Verticillium wilt, a soilborne vascular disease caused by Verticillium dahliae, strongly affects cotton yield and quality. In this study, an isolated rhizosphere bacterium, designated Bacillus velezensis BvZ45-1, exhibited greater than 46% biocontrol efficacy against cotton verticillium wilt under greenhouse and field conditions. Moreover, through crude protein extraction and mass spectrometry analysis, we found many antifungal compounds present in the crude protein extract of BvZ45-1. The purified oxalate decarboxylase Odx_S12 from BvZ45-1 inhibited the growth of V. dahliae Vd080 by reducing the spore yield, causing mycelia to rupture, and causing spore morphology changes, cell membrane rupture and cell death. Subsequently, overexpression of Odx_S12 in Arabidopsis significantly improved plant resistance to V. dahliae. Through studies of the resistance mechanism of Odx_S12, V. dahliae was shown to produce oxalic acid (OA), which has a toxic effect on Arabidopsis leaves. Odx_S12 overexpression reduced Arabidopsis OA content, enhanced tolerance to OA, and improved resistance to verticillium wilt. Transcriptome analysis and quantitative real-time PCR (qRT-PCR) analysis revealed that Odx_S12 promoted the outbreak of reactive oxygen species (ROS) and a salicylic acid (SA) and abscisic acid (ABA) mediated defense response in Arabidopsis. In summary, this study not only identified B. velezensis BvZ45-1 as an efficient biological control agent, but also identified the resistance gene Odx_S12 as a candidate for cotton breeding against verticillium wilt.

3D-printed nanohydroxyapatite/methylacrylylated silk fibroin scaffold for repairing rat skull defects

The repair of bone defects remains a major challenge in the clinic, and treatment requires bone grafts or bone replacement materials. Existing biomaterials have many limitations and cannot meet the various needs of clinical applications. To treat bone defects, we constructed a nanohydroxyapatite (nHA)/methylacrylylated silk fibroin (MASF) composite biological scaffold using photocurable 3D printing technology. In this study, scanning electron microscopy (SEM) was used to detect the changes in the morphological structure of the composite scaffold with different contents of nanohydroxyapatite, and FTIR was used to detect the functional groups and chemical bonds in the composite scaffold to determine the specific components of the scaffold. In in vitro experiments, bone marrow mesenchymal stem cells from SD rats were cocultured with scaffolds soaking solution, and the cytotoxicity, cell proliferation, Western blot analysis, Quantitative real-time PCR analysis, bone alkaline phosphatase activity and alizarin red staining of scaffolds were detected to determine the biocompatibility of scaffolds and the effect of promoting proliferation and osteogenesis of bone marrow mesenchymal stem cells in vitro. In the in vivo experiment, the skull defect was constructed by adult SD rats, and the scaffold was implanted into the skull defect site. After 4 weeks and 8 weeks of culture, the specific osteogenic effect of the scaffold in the skull defect site was detected by animal micro-CT, hematoxylin and eosin (HE) staining and Masson's staining. Through the analysis of the morphological structure of the scaffold, we found that the frame supported good retention of the lamellar structure of silk fibroin, when mixed with nHA, the surface of the stent was rougher, the cell contact area increased, and cell adhesion and lamellar microstructure for cell migration and proliferation of the microenvironment provided a better space. FTIR results showed that the scaffold completely retained the β -folded structure of silk fibroin, and the scaffold composite was present without obvious impurities. The staining results of live/dead cells showed that the constructed scaffolds had no significant cytotoxicity, and thw CCK-8 assay also showed that the constructed scaffolds had good biocompatibility. The results of osteogenic induction showed that the scaffold had good osteogenic induction ability. Moreover, the results also showed that the scaffold with a MASF: nHA ratio of 1: 0.5 (SFH) showed better osteogenic ability. The micro-CT and bone histometric results were consistent with the in vitro results after stent implantation, and there was more bone formation at the bone defect site in the SFH group.This research used photocurable 3D printing technology to successfully build an osteogenesis bracket. The results show that the constructed nHA/MASF biological composite material, has good biocompatibility and good osteogenesis function. At the same time, in the microenvironment, the material can also promote bone defect repair and can potentially be used as a bone defect filling material for bone regeneration applications.

Key anti-freeze genes and pathways of Lanzhou lily (Lilium davidii, var. unicolor) during the seedling stage.

Temperature is one of the most important environmental factors for plant growth, as low-temperature freezing damage seriously affects the yield and distribution of plants. The Lanzhou lily (Lilium davidii, var. unicolor) is a famous ornamental plant with high ornamental value. Using an Illumina HiSeq transcriptome sequencing platform, sequencing was conducted on Lanzhou lilies exposed to two different temperature conditions: a normal temperature treatment at 20°C (A) and a cold treatment at -4°C (C). After being treated for 24 hours, a total of 5848 differentially expressed genes (DEGs) were identified, including 3478 significantly up regulated genes and 2370 significantly down regulated genes, accounting for 10.27% of the total number of DEGs. Quantitative real-time PCR (QRT-PCR) analysis showed that the expression trends of 10 randomly selected DEGs coincided with the results of high-throughput sequencing. In addition, genes responding to low-temperature stress were analyzed using the interaction regulatory network method. The anti-freeze pathway of Lanzhou lily was found to involve the photosynthetic and metabolic pathways, and the key freezing resistance genes were the OLEO3 gene, 9 CBF family genes, and C2H2 transcription factor c117817_g1 (ZFP). This lays the foundation for revealing the underlying mechanism of the molecular anti-freeze mechanism in Lanzhou lily.

Genome-Wide Analysis of the Xyloglucan Endotransglucosylase/Hydrolase (XTH) Gene Family: Expression Pattern during Magnesium Stress Treatment in the Mulberry Plant (Morus alba L.) Leaves.

Mulberry (Morus alba L.), a significant fruit tree crop, requires magnesium (Mg) for its optimal growth and productivity. Nonetheless, our understanding of the molecular basis underlying magnesium stress tolerance in mulberry plants remains unexplored. In our previous study, we identified several differential candidate genes associated with Mg homeostasis via transcriptome analysis, including the xyloglucan endotransglucosylase/hydrolase (XTH) gene family. The XTH gene family is crucial for plant cell wall reconstruction and stress responses. These genes have been identified and thoroughly investigated in various plant species. However, there is no research pertaining to XTH genes within the M. alba plant. This research systematically examined the M. alba XTH (MaXTH) gene family at the genomic level using a bioinformatic approach. In total, 22 MaXTH genes were discovered and contained the Glyco_hydro_16 and XET_C conserved domains. The MaXTHs were categorized into five distinct groups by their phylogenetic relationships. The gene structure possesses four exons and three introns. Furthermore, the MaXTH gene promoter analysis reveals a plethora of cis-regulatory elements, mainly stress responsiveness, phytohormone responsiveness, and growth and development. GO analysis indicated that MaXTHs encode proteins that exhibit xyloglucan xyloglucosyl transferase and hydrolase activities in addition to cell wall biogenesis as well as xyloglucan and carbohydrate metabolic processes. Moreover, a synteny analysis unveiled an evolutionary relationship between the XTH genes in M. alba and those in three other species: A. thaliana, P. trichocarpa, and Zea mays. Expression profiles from RNA-Seq data displayed distinct expression patterns of XTH genes in M. alba leaf tissue during Mg treatments. Real-time quantitative PCR analysis confirmed the expression of the MaXTH genes in Mg stress response. Overall, this research enhances our understanding of the characteristics of MaXTH gene family members and lays the foundation for future functional genomic study in M. alba.

Genome-Wide Identification of PYL/RCAR ABA Receptors and Functional Analysis of LbPYL10 in Heat Tolerance in Goji (Lycium barbarum).

The characterization of the PYL/RCAR ABA receptors in a great deal of plant species has dramatically advanced the study of ABA functions involved in key physiological processes. However, the genes in this family are still unclear in Lycium (Goji) plants, one of the well-known economically, medicinally, and ecologically valuable fruit crops. In the present work, 12 homologs of Arabidopsis PYL/RCAR ABA receptors were first identified and characterized from Lycium (L.) barbarum (LbPYLs). The quantitative real-time PCR (qRT-PCR) analysis showed that these genes had clear tissue-specific expression patterns, and most of them were transcribed in the root with the largest amount. Among the three subfamilies, while the Group I and Group III members were down-regulated by extraneous ABA, the Group II members were up-regulated. At 42 °C, most transcripts showed a rapid and violent up-regulation response to higher temperature, especially members of Group II. One of the genes in the Group II members, LbPYL10, was further functionally validated by virus-induced gene silencing (VIGS) technology. LbPYL10 positively regulates heat stress tolerance in L. barbarum by alleviating chlorophyll degradation, thus maintaining chlorophyll stability. Integrating the endogenous ABA level increase following heat stress, it may be concluded that LbPYL-mediated ABA signaling plays a vital role in the thermotolerance of L. barbarum plants. Our results highlight the strong potential of LbPYL genes in breeding genetically modified L. barbarum crops that acclimate to climate change.

Targeting osteopontin alleviates endometriosis and inflammation by inhibiting the RhoA/ROS axis and achieves non-invasive in vitro detection via menstrual blood.

How does osteopontin (OPN) in endometriosis ectopic stromal cells (EESCs) participate in the pathogenesis of endometriosis and achieve non-invasive detection in vitro? Targeted OPN regulates endometriosis's necroptosis and inflammatory state by inhibiting the RhoA/reactive oxygen species (ROS) axis, thereby alleviating endometriosis and enabling non-invasive detection of menstrual blood in vitro. Endometriosis is a chronic inflammatory disease. Recent studies have shown that OPN plays an important role in disease progression by regulating cell death and inflammation. The study included 20 patients diagnosed with endometriosis (confirmed by laparoscopy and histology) and 10 controls without endometriosis. Endometriotic stromal cells were isolated from endometrial samples, while menstrual blood endometrial cells (MESCs) were isolated from menstrual blood. These cells were then cultured in vitro and utilized in subsequent experiments. OPN expression in EESCs was assessed using inflammatory factor sequencing, immunohistochemical staining (IHC), quantitative real-time PCR (qRT-PCR) analysis, and Western blotting (WB). The biological behavior of OPN and its effects on inflammatory factors were examined using EdU, wound-healing, Transwell, and ELISA assays. Necroptosis in EESCs and its impact on inflammatory factors were detected through qRT-PCR, WB, and Calcein-AM/PI fluorescence assays. The examination of mitochondrial stress in EESCs involved the use of the Mitochondrial Membrane Potential (ΔΨm) Assay, ROS detection, and Calcein-AM Loading/cobalt chloride Quenching. qRT-PCR, WB, and other experiments were conducted to verify the regulation of necroptosis and inflammatory factor levels in EESCs by OPN through the RhoA/ROS axis. Knockdown of OPN and its inhibitory effect on endometriosis lesion size were confirmed using AAV9 virus, IHC, qRT-PCR, WB, and other experiments. Additionally, OPN expression in MESCs was detected using transcriptome sequencing, RT-PCR, WB, and other experiments. In vitro assays demonstrated a significant upregulation of OPN in EESCs, and the knockdown of OPN effectively inhibited necroptosis and the release of inflammatory factors. OPN inhibited necroptosis and inflammatory factor release by mediating RhoA-dependent ROS production and blocking mixed lineage kinase domain-like protein phosphorylation at the cell membrane. In vivo, targeting of OPN can inhibit the growth of endometriosis lesions. Clinically, OPN was also significantly upregulated in the menstrual blood of patients with endometriosis. N/A. Due to limitations in obtaining surgical specimens, our study primarily involved collecting endometriosis tissues from women during the proliferative and secretory phases of the menstrual cycle. We observed a significant overexpression of OPN in the samples used for our investigation. However, the expression of OPN in endometriosis tissues during the intermenstrual phase remains unknown. Our findings highlight the pivotal role of the OPN/RhoA/ROS axis in the regulation of necroptosis and the release of inflammatory factors. OPN knockdown exerts a therapeutic effect in vivo, and the high expression detection of OPN in menstrual blood in vitro. In summary, targeting OPN provides possibilities for the treatment and detection of endometriosis. This study was supported by the National Natural Science Foundation of China (82071626), the Zhejiang Province Public Welfare Technology Application Research Project (LGF21H040010), and the Clinical Research project of the Second Affiliated Hospital of Wenzhou Medical University (1010293). The authors have no conflicts of interest.

Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies.

Sugar transporters play important roles in plant growth and development, flowering and fruiting, as well as responses to adverse abiotic and biotic environmental conditions. Lilies (Lilium spp.) are some of the most representative ornamental bulbous flowers. Sugar metabolism is critical for bulb formation in lilies; therefore, clarifying the amount and expression pattern of sugar transporters is essential for further analyzing their roles in bulb formation. In this study, based on the transcriptome data of the Lilium Oriental hybrid 'Sorbonne' and Lilium × formolongi, a total of 69 and 41 sugar transporters were identified in 'Sorbonne' and Lilium × formolongi, respectively, by performing bioinformatics analysis. Through phylogenetic analysis, monosaccharide transporters (MSTs) can be divided into seven subfamilies, sucrose transporters (SUTs) can be divided into three subgroups, and sugars will eventually be exported transporters (SWEETs) can be divided into four clades. According to an analysis of conserved motifs, 20, 14, and 12 conserved motifs were predicted in MSTs, SUTs, and SWEETs, respectively. A conserved domain analysis showed that MSTs and SUTs contained a single domain, whereas most of the SWEETs harbored two MtN3/saliva domains, also known as a PQ-loop repeat. The LohINT1, which was predicted to have a smaller number of transmembrane structural domains, was cloned and analyzed for subcellular localization. It was found that the LohINT1 protein is mainly localized in the cell membrane. In addition, the expression analysis indicated that 22 LohMSTs, 1 LohSUTs, and 5 LohSWEETs were upregulated in 'Sorbonne' 1 day after scale detachment treatment, suggesting that they may regulate the initiation of the bulblet. A total of 10 LflMSTs, 1 LflSUTs, and 6 LflSWEETs were upregulated 4~6 months after sowing, which corresponds to the juvenile-to-adult transition phase of Lilium × formolongi, suggesting that they may also play a role in the accompanying bulb swelling process. Combined with quantitative real-time PCR (qRT-PCR) analysis, LohSTP8 and LohSTP12 were significantly overexpressed during the extremely early stage of bulblet initiation, and LflERD6.3 was significantly overexpressed during the growth of the underground bulblet, suggesting that they may be key sugar transporters in the formation of lily bulbs, which needs further functional verification.

Impacts of 1-methylcyclopropene (1-MCP) and ethephon treatments on expression of aquaporin (AQP) genes during apple fruit ripening

The main aim of the present study was to determine the effects of ethephon (ET) and 1-methylcyclopropene (1-MCP) treatments on expression of aquaporin (AQP) genes during apple fruit ripening. The harvested apples were treated with 1-MCP (1 μL L−1) and ET (0.2 mmol L−1), respectively, and then ripened at room temperature (20 ± 1 °C). Our results suggested that 1-MCP application reduced weight loss, total soluble solids (TSS) content, respiration rate and ethylene production, maintained flesh firmness, and slowed down the increase in content of soluble sugar and the decrease in content of titratable acidity (TA) and dry matter concentration (DMC) in flesh and peel during ripening compared with the control, while ET treatment had the opposite effects. Quantitative real-time PCR (qPCR) analysis indicated that MdAQPs expression was differentially regulated by both treatments in flesh and peel during ripening. Expression of MdSIP1;2 and MdNIP5;3 in flesh and MdPIP1;3 and MdPIP2;7 in peel was generally up-regulated by both treatments, while MdTIP4;2 expression in peel was down-regulated by both treatments during ripening. Expression of MdNIP1;1, MdNIP2;1 and MdTIP1;1 in flesh and MdTIP2;2, MdTIP3;1 and MdNIP5;1 in peel was differentially down-regulated by ET treatment while differentially up-regulated by 1-MCP application during ripening. Transcript levels of MdTIP2;2, MdPIP1;2, MdPIP2;4, MdPIP2;3 and MdNIP5;2 in flesh and MdNIP2;1, MdSIP2;2, MdSIP1;2 and MdNIP1;1 in peel were differentially up-regulated by ET application while differentially reduced by 1-MCP application during ripening. After transient over-expression of MdERF2, MdAQPs exhibited varying expression profiles in both tissues during ripening. The results indicated that MdAQPs expression was modulated by ethylene signal in ripening apple fruit.